Protein nanopore devices have been explored for many sensor applications, especially in the field of DNA/RNA sequencing. Discrimination of nucleotide bases has been demonstrated by reading the ionic current signal when the DNA molecule is passing through the nanopore. Following from these initial observations, protein based nanopore devices have been postulated to have great potential to push forward nucleic acid sequencing technology in terms of lower price, fast turn around and longer read length.
A more mature technology, sequencing-by synthesis (SBS), has made rapid progress in the last five years, doubling throughput about every six months, far outpacing Moore's law for the semiconductor industry. This rapid progress has been enabled by primarily three key improvements to SBS protocols: (1) cycle sequencing chemistry improvements; (2) increases in density of nucleic acid colonies on surfaces; and (3) improvement in imaging/scanning technology. These three technological improvements have increased throughput of commercial systems from about 1 Gb per run in January 2007 to over 1 Tb per run in June 2012. However, these technological advances have not been found to be directly portable to nanopore technologies.
In spite of this rapid progress in SBS improvement, 30X genome prices are still over $1000/genome with turn-around times in excess of a week. Moreover, de novo assembly and haplotyping of human genomes obtained via SBS is challenged by short reads. Strand sequencing via nanopores, can potentially read up to 50,000 bases within a few minutes. This single molecule platform to date appears to achieve speed but at the cost of greatly decreased parallelization.
Parallelization of biological nanopores is notoriously difficult. The fragility of the platform itself, especially the semi-fluidic nature of the lipid bilayer, demands specialized handling often by highly trained technicians, making nanopore systems less practical for wide spread commercialization. The current technique for assembly of protein nanopore devices is mostly manual, laborious and time consuming. After painting a lipid bilayer over a substrate having an array of micrometer sized apertures, the operator contacts the coated array with a solution having a carefully titrated quantity of the protein. The amount of nanopore is selected according to Poisson statistics to maximize the number of apertures that acquire a nanopore while minimizing the number of apertures that are loaded with greater than one nanopore. After a specific incubation period has lapsed, the nanopore-containing solution is washed away. Poisson loading requires the time period to be carefully selected to achieve the highest possible number of apertures having one and only one nanopore. The results are highly dependent upon the skill of the operator and not easily adaptable to large scale device manufacturing.
Thus, a major challenge for nanopore technology is to increase the robustness of the platform and simultaneously improve parallelization, for example, in sequencing applications. The present disclosure addresses this need and provides other advantages as well.